Plant BIO 2017-12-14 cellular respiration full lab report PDF

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1 Sarah Long Plant Biology Dr. Gagnon 14 December 2017 Cellular Respiration Abstract In this experiment, succinate was converted to fumarate through cellular respiration that was catalyzed by DPIP. Different concentrations of DPIP were tested to determine which is the optimum amount and concentration for an efficient reaction. The absorbance of each was measured with a Spec-20. Our results indicated that absorbance increases along with concentration and amount of DPIP used, leading us to the conclusion that DPIP is a strong catalyst for the reaction and higher levels promote a more efficient reaction. Introduction Aerobic respiration is used by most living things to produce ATP because it is the most efficient form of energy synthesis and successfully extracts most of the energy stored in the bonds of glucose molecules, when compared to fermentation which does not extract as much usable energy. Alcoholic fermentation has a yield of two ATP molecules per glucose molecule, while aerobic respiration has a yield of 38 ATP molecules per glucose molecule. Aerobic respiration begins with glycolysis, followed by the Krebs cycle. The Krebs cycle releases electrons, which then enter the electron transport chain; the electron transport chain is what produces the ATP. Glycolysis occurs in the cytoplasm and the Krebs cycle and the electron transport chain occur in the mitochondrion. In this lab, a suspension of pulverized lima beans containing mitochondria was used to perform aerobic respiration the way that full cells perform it. Sucrose serves as the glucose source in the experiment. An enzyme was used to catalyze the conversion of succinate to fumarate, and DPIP intercepted the electrons before they entered the electron transport chain. This makes its color change from blue to clear – when the solution is clear, it signals that the reaction has been completed. Materials and methods For this experiment, soaked lima beans were cut into pieces and ground in a blender in 25 mL of assay buffer for two minutes. The mixture was poured through four layers of cheesecloth and the filtrate was collected in a beaker. 10 more mL of buffer was used to rinse the blender and the blender was filtered through the cheesecloth as well. The filtrate was chilled over ice for 10 minutes. A microtube was filled with 1.5 mL of filtrate and placed in the microcentrifuge, and then centrifuged for at least 5 minutes at 2000 rpm. The supernatant was transferred to another tube, and 1.5 mL of cold sucrose/phosphate buffer was added to the pellet before the pellet material was resuspended. The Spec-20 was turned on and allowed to warm up for 20 minutes. The wavelength was set to 600 nm and the mode was set to transmittance. The transmittance knob was turned to the

2 left until the reading was 0. The mode was then set to absorbance and a blank was placed into the chamber. The white line on the cuvette was lined up with the arrow on the Spec-20. The lid was closed and the front right knob was turned until the reading was 0. 6 cuvettes were labeled #1-6. DPIP and sucrose/phosphate buffers were added to the cuvettes in the amounts and concentrations specified in Table 1 below. Tube #1 was used as a blank and the Spec-20 was zeroed for absorbance. The absorbance at 600 nm was recorded for each concentration of DPIP. 4 cuvettes were labeled #1-4 and solution was added to each tube in order. Tubes 1 and 3 were zero tubes – the machine was zeroed every two minutes. Results Table 1: absorbance at 600 nm vs concentrations and different amounts of DPIP and amount of assay buffers. Tube #

Conc. of DPIP Amount of DPIP (mL) 0% 0 2% 0.1 4% 0.2 8% 0.4 12% 0.6 16% 0.8

1 2 3 4 5 6

Amount of assay buffer (mL) 5.0 4.9 4.8 4.6 4.4 4.2

Absorbance at 600 nm 0.000 0.113 0.202 0.309 0.500 0.640

Figure 1: absorbance at 600 nm vs concentration of DPIP.

Absorbance at 600 nm 0.7 0.6 Absorbance

0.5 0.4 0.3 0.2 0.1 0 0%

2%

4%

8%

Concentration of DPIP

Figure 2: absorbance vs time.

12%

16%

3

Absorbance vs time Absorbance (600 nm)

0.5 0.4 0.3 Tube 2 Tube 4

0.2 0.1 0 0

2

4

6

8

10

12

14

16

18

20

-0.1 Time (min)

Discussion and conclusions As shown in Table 1 and Figures 1 and 2 above, absorbance increased as concentration of DPIP increased. This further proves the hypothesis that DPIP is an essential catalyst for the reaction that converts succinate to fumarate. The more DPIP added to the cuvette and the higher the concentration of DPIP in the cuvette, the greater the absorbance at a wavelength of 600 nm. Enzyme activity was measured with DPIP. The compound changes color from blue to clear as the reaction occurs, so a clear and colorless solution signals a completed reaction and the end of enzyme activity. The process of the color changing is indicative of the enzymes working properly to catalyze the reaction. Based on our results, cellular respiration occurs in the mitochondria – the mitochondria, in this case, were present in the ground lima bean mixture prepared at the beginning of the experiment. The lima beans stand in for real cells, and the process was carried out as it normally is in healthy living cells. If the electron transport chain broke down, the cell would produce no ATP through cellular respiration: the electron transport chain is responsible for the actual production of ATP. If this were the case, the cell would have to resort to fermentation to produce ATP, because fermentation uses glycolysis but not the Krebs cycle or electron transport chain and therefore would still be able to synthesize ATP if the chain broke down. However, cellular respiration has a yield of 38 ATP molecules per glucose molecule while fermentation only has a yield of 2 ATP molecules per glucose molecule. For this reason cellular respiration is a much more efficient energy-producing process than fermentation. Sodium azide can wreak havoc on a living organism’s systems. It stimulates the synthesis of excitatory amino acids and therefore drastically lowers blood pressure and can induce convulsions. It is considered highly toxic to living things. Azide anions can also inhibit catalase, peroxidase, and cytochrome oxidase, all vital for living systems. Releasing small amounts of energy in a series of steps rather than a large amount all at once allows the cell to conserve energy. When it is released in small steps, it is used in small

4 steps – when released all at once, the cell can burn through energy quickly and then run out before it has the chance to synthesize more. For this reason, it is much more energy efficient to produce and release molecules such as ATP gradually and slowly....